The effect of alpha1-adrenergic stimulation on the inotropy of the myocardium is still controversial. A positive inotropic effect has ben well documented and it is often preceded by a short lived decrease in contractility. Additionally a persistent negative inotropic effect of alpha1 stimulation has also been described in multicellular cardiac preparations. The purpose of this study was to characterized the effect of alpha1 stimulation on the cytosolic Ca2+ (Cai) transient and on the contractile properties of single ventricular myocytes under condition leading either to an enhancement or a decrease in twitch amplitude. Isolated rat ventricular myocytes, pretreated with propranolol were used to investigate the effect of alpha1-adrenergic stimulation with phenylephrine on twitch amplitude (TA) and on the frequency of spontaneous contractile waves (CW), which represent the mechanical expression of spontaneous Ca2+ release from the sarcoplasmic reticulum (SR). In 1 mM bathing (Ca2+) (Cao), during field stimulation at 0.2 Hz, alpha1 had a positive inotropic effect which was reversibly abolished by prazosin and to a considerable extent appeared related to an increased myofilament responsiveness to Ca2+ in addition to some enhancement in the amplitude of the Cai transient associated with the twitch. b. During stimulation at 0.2 hz, in d mM Cao, CW appeared in some of the diastolic intervals. Phenylephrine abolished them and decreased TA. This negative effects was in contrast to the positive action observed in parallel studies in 1 mM Cao. In similar experiments with myocytes loaded with the Ca2+ probe Indo-1 AM the negative effect of alpha1 on TA was associated with a decrease in the amplitude of the Cai transient. c. In the absence of stimulation, in d mM Cao, alpha1 significantly and reversibly reduced CW frequency. This effect could be prevented by the alpha1 blocker prazosin. These findings are consistent with the view that the effect of alpha1-adrenergic stimulation of cardiac cells can vary in relation to intracellular (Ca2+).